Blood typing, and particularly the determination of the presence or absence of Rh (D) antigen, is a routine procedure in modern medicine. Since the discovery of the relationship between Rh factor and disease in the 1930's, there has been an increasing concern to detect Rh incompatibilities between mother and fetus so that such incompatibilities may be treated or avoided in future children. At the present time, many state laws require Rh testing of pregnant women and of infants born of Rh negative women. The introduction of Rh immune globulin in 1968 allowed for the first time a method of treatment of Rh negative women bearing Rh positive children to prevent disease in later Rh positive children of these women.
The United States Food and Drug Administration (FDA) requires that an anti-D antiserum have a potency titer value at least equal to that of the FDA reference serum, as set out in a chapter entitled "Performance Criteria For Blood Grouping Sera" authored by P. A. H. Hoppe from the Immunohematology Branch, Division of Blood and Blood Products, Bureau of Biologics, Food and Drug Administration. Appendix I of this publication presents observed titer values of the anti-D (slide and rapid tube) FDA reference serum, which vary from twenty-nine to fifty-eight, depending upon the test call phenotype used. In other words, the reference serum gave a reaction rated as "one plus" at dilutions varying from 1:32 to 1:64 (titers of 32 to 64). This appendix I demonstrates that an anti-D product meets the FDA potency requirements if it has an average titer of at least 32 for Dce cells in the slide tests or rapid tube test.
There are two types of diagnostic tests currently used for detecting the presence of D antigen. It should be understood, by way of introduction, that IgG antibody to D antigen is a so-called "incomplete" antibody. That is, IgG antibodies with specificity for the D antigen often fail to agglutinate Rh positive red cells suspended in saline. In contrast, IgM antibodies to the D antigen do cause agglutination of Rh positive red cells in saline.
The first of these tests uses IgG anti-D antibody as the reagent. Since anti-D IgG will not by itself cause agglutination of Rh positive red blood cells in saline, some potentiating means must be found to cause the agglutination. This potentiation is accomplished by a relatively high concentration of a biological polymer (e.g., nonspecific protein such as albumin) or a synthetic polymer (e.g., polyvinylpyrrolidone or a dextran). Typically, the concentration of albumin in the final reagent composition is approximately 20 to 25 percent. While this added protein succeeds in potentiating the agglutination of specific IgG anti-D antibodies and red blood cells having D antigen, it also causes nonspecific agglutination of red blood cells having IgG coated on the surface thereof. Such coating occurs, for example, in individuals having such diseases as autoimmune hemolytic anemia or hemolytic disease of the newborn, in which antibody is produced to the patient's own red blood cells and binds thereto. False positive results for presence of Rh antigen will therefore be obtained for these individuals with this test. One significant advantage of this test, however, is its rapidity, since little (if any) incubation is required prior to reading the results. This test may be conducted in either a test tube or a glass slide. A second advantage is the ability to detect certain weak antigens denominated D.sup.u, as further described below.
The second current test for detection of D antigen uses IgM antibody instead of IgG antibody. Since IgM is a complete antibody with respect to the D antigen, the addition of a potentiator is not required; typically, the reagent composition contains a total protein concentration of less than about 8% by weight, accomplished by adding (e.g.) bovine albumin to the IgM antiserum. This test can be conducted only in a test tube. While this test removes the nonspecific agglutination of IgG coated cells which occurs with the potentiated IgG test, it also suffers from several disadvantages. The principal disadvantages are that the test requires a fifteen to sixty minute incubation at 37.degree. C., which step is not required by the potentiated IgG test, and also that it requires IgM antibody, which is rare and difficult to obtain. Additionally, it is not possible to run the so-called D.sup.u test in conjunction with this second type of test, since this D.sup.u test uses antibody to IgG as its reagent. This reagent antibody obviously does not react with IgM.
Thus, if one could develop a rapid test employing IgG antibody in a low protein formulation, a long felt need would be satisfied.